Are the plates made of the same material? The loss of signal could be due to the ANS binding to the plate surface. Polystyrene plates are more likely to have this problem than polypropylene plates.

Another potential reason for the observation could be a difference between the two plate types in regard to a change in the height of the liquid in the wells as the sample evaporates over time.

Thanks!  Ordering some samples of polypropylene plates and going to try again, as I'm not sure what our old plates were made of.  Appreciate that!

I would stay away from V-bottom wells for any light-based plate assays.  The plate-reading instruments are engineered for flat bottom wells, and I would worry that the V-bottom well would introduce light scattering and/or reflections that could be highly problematic for accurate detection.  The degree of negative impact will depend a lot on specific instrument design, but I think there are plenty of reasons to just avoid V-bottom wells for this type of assay.

Thank you for this input.  We had previously done the assay in a fluorospectrometer, which was tedious as we had to test each ligand individually.  When I tried in the Tecan, I repeated some of my previous experiments with v-bottom plates and got basically the same results.  So I thought everything was kosher.  But I did not know that the instrument was engineered for flat bottom wells (and actually the plate setting I was using was for flat-bottom since there was no V-bottom option.)  So it appears I have some more work to do....

The reason for V bottom well is to allow the light to have sufficient pathlength compared to flat bottom well. When you have very smaller volumes, it is ideal to use V shaped wells for snaller volumes so that you can get better pathlength and greater sensitivity